We have continued to use our Friend spleen focus-forming virus (SFFV)-induced mouse model for leukemia to understand the molecular changes associated with cancer as well as to test the efficacy of pharmacological interventions. Our previous studies have shown that the erythroleukemia caused in mice by the retrovirus Friend spleen focus-forming virus (SFFV) is the result of activation of the receptor tyrosine kinase short-form Stk (sf-Stk) by the viral envelope protein, which interacts with sf-Stk to cause constitutive activation of signal transduction pathways needed for erythropoietin-independent erythroid cell hyperplasia. We also showed that expression of SFFV-activated sf-Stk in rodent fibroblasts results in their transformation. Recent studies in the literature have suggested that the human counterpart of sf-Stk, the receptor tyrosine kinase short-form Ron (sf-Ron), plays a role in promoting cancer in humans. Although drugs have been designed to target the kinase domain of sf-Ron and other tyrosine kinases, treatment with such drugs often leads to resistance due to secondary mutations of the kinase. In collaboration with Kazuo Nishigaki, we, therefore, sought to identify an alternative target for inhibiting transformation by sf-Ron. Our previous studies with the mouse tyrosine kinase sf-Stk indicated that it could transform rodent fibroblasts when it was co-expressed with the SFFV envelope protein and that the extracellular domain of sf-Stk, which interacts with the viral envelope protein, was critical for this process. We have now shown that human sf-Ron can also transform rodent fibroblasts but, unlike transformation by mouse sf-Stk, co-expression with the SFFV envelope protein is not required. However, chimeras between sf-Ron and sf-Stk indicate that the extracellular domain of sf-Ron is still required for its ability to transform fibroblasts. Deletion and mutational analysis of the extracellular domain of sf-Ron revealed amino acid residues critical for induction of fibroblast transformation, and serial deletion mutants from the N-terminal region of sf-Ron suggested that structural conformation was associated with the ability of the kinase to transform fibroblasts. These results indicate that fibroblast transformation by sf-Ron is dependent on the structure of its extracellular domain, which may bind a cellular protein, and suggest that therapeutic interventions targeting this domain could be effective in blocking the biological effects of this human tyrosine kinase. We have also used our retrovirus-induced mouse model for leukemia to study a promising anti-cancer agent, the nitric oxide prodrug JS-K. In collaboration with the CCR laboratory of Larry Keefer, we showed that JS-K, which consists of a diazeniumdiolate group necessary for the release of NO as well as an arylating ring, was cytotoxic for SFFV-transformed murine erythroleukemia (SFFV-MEL) cells at a low IC50 value. We subsequently carried out studies to understand the mechanism by which JS-K kills the murine erythroleukemia cells and to determine the roles of NO and arylation in the process. Our studies indicate that JS-K inhibits the PI 3-kinase/Akt and MAP kinase pathways. This correlates with the activation of the tumor suppressor FoxO3a and increased expression of the cyclin-dependent kinase inhibitor p27Kip1 and various caspases, leading to cell cycle arrest and apoptosis. The arylating capability of JS-K appears to be sufficient for inducing these biological effects since AcOM-DEA/NO, an NO donor without arylating capacity, was ineffective at killing SFFV-MEL cells and the NO quencher vitamin B12 failed to significantly diminish the cytotoxicity of JS-K towards the transformed erythroid cells. Overall, these data suggest that JS-K kills murine erythroleukemia cells by arylating and inactivating signaling molecules that block the activation of a tumor suppressor. Finally, we have continued to use our retrovirus-induced rat model for neurodegenerative disease to test pharmacological interventions based on our studies to uncover the biological changes associated with the development of neurodegeneration. We previously demonstrated that PVC-211 MuLV infection of brain capillary endothelial cells results in the production of vascular endothelial cell growth factor (VEGF) and the chemokine macrophage inflammatory protein-1 (MIP-1) alpha, leading to vascular leakage and activation of microglia. Further studies demonstrated that depletion of microglia from rat brains blocks neurodegeneration induced by PVC-211 MuLV and that treatment with antiserum to MIP-1 alpha or splenectomy, both of which reduce the number of activated microglia in the brain, can delay disease, clearly demonstrating the importance of activated microglia in the development of PVC-211 MuLV-induced neurodegeneration. Since the production of VEGF is one of the earliest events occurring in our rat model of neurodegeneration, we are currently testing pharmacological inhibitors of VEGF for their ability to block or mitigate PVC-211 MuLV-induced neurodegeneration.